Microscope system

ABSTRACT

A microscope system as an optical microscope system for observing a specimen includes: an imaging optical system that forms an image of transmitted light or reflected light from the specimen; an illumination light source that illuminates illumination light on the specimen; an illumination optical system that has a first spatial light modulation element, which changes intensity distribution of the illumination light at a conjugate position of a pupil of the imaging optical system, and illuminates light, which is originated from the illumination light source, on the specimen; an image sensor that detects light through the imaging optical system; and a calculation section that calculates the intensity distribution of the illumination light appropriate for observation of the specimen on the basis of the intensity distribution of the illumination light formed by the first spatial light modulation element and output data detected by the image sensor.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a non-provisional application claiming priority toand the benefit of U.S. provisional application No. 61/534,197, filed onSep. 13, 2011, and claims priority to Japanese Patent Application No.2010-235155, filed on Oct. 20, 2010. The entire contents of which areincorporated herein by reference.

BACKGROUND

1. Field of the Invention

The present invention relates to a microscope system that derives andforms intensity distribution of illumination light appropriate forobservation.

2. Description of Related Art

In bright field microscopes, the intensity distribution of theillumination light is adjusted by changing a diaphragm having a circularshape. Furthermore, the shape of the diaphragm may be selected andapplied on the basis of determination of an observer. In phase-contrastmicroscopes, a ring diaphragm and a phase ring form the intensitydistribution of the illumination light.

Since the intensity distribution of the illumination light has a greateffect on an observational image of a specimen, a circular diaphragm, aring diaphragm and a phase ring, or the like have been subjected totests to further improve the observation picture of the specimen. Forexample, in Japanese Unexamined Patent Application Publication No.2009-237109, a modulation section is provided to surround a ring regionwhich is formed in a ring shape of the phase ring, and is formed suchthat the direction of the transmission axis of the modulation section isdifferent from that of a region other than the modulation section,thereby embodying a phase-contrast microscope capable of continuouslychanging the contrast.

SUMMARY

However, in the above-mentioned microscopes, the shape of the diaphragmis fixed to some extent, and there is a limitation in adjustment of theintensity distribution of the illumination light. Furthermore, even in acase of selecting the shape of the diaphragm, the selection is performedon the basis of determination or experience of the observer, and thusthe shape of the diaphragm is not always formed to be able to observethe image of the object at its best condition during observation.Furthermore, in the phase-contrast microscopes, the positions of thering diaphragm and the phase ring are fixed, and thus it is difficult tofreely select the shape and observe the image of the object at its bestcondition during the observation.

Accordingly, the present invention provides a microscope system thatderives and forms the intensity distribution of the illumination lightappropriate to observe the specimen.

According to a first aspect, a microscope system as an opticalmicroscope system for observing a specimen includes: an imaging opticalsystem that forms an image of transmitted light or reflected light fromthe specimen; an illumination light source that illuminates illuminationlight to the specimen; an illumination optical system that has a firstspatial light modulation element, which changes the intensitydistribution of the illumination light at a conjugate position of apupil of the imaging optical system, and illuminates light, which isoriginated from the illumination light source, on the specimen; an imagesensor that detects light through the imaging optical system; and acalculation section that calculates the intensity distribution of theillumination light appropriate for observation of the specimen on thebasis of the intensity distribution of the illumination light formed bythe first spatial light modulation element and output data detected bythe image sensor.

According to the present invention, there is provided a microscopesystem that derives and forms the intensity distribution of theillumination light appropriate to observe an image of an object in goodcondition during the observation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic configuration diagram of a microscope system 100.

FIG. 2A is a schematic configuration diagram in a case where a firstspatial light modulation element 90 is a liquid crystal panel 93. FIG.2B is a schematic configuration diagram in a case where the firstspatial light modulation element 90 is a digital micro mirror device(DMD) 94.

FIG. 3 is a flowchart of the hill climbing method that finds anappropriate illumination region 91.

FIG. 4A is a diagram of a region setting portion 22 and a parametersetting portion 23 of a display section 21.

FIG. 4B is a diagram of the display section 21 in a case of setting theillumination region 91 of the first spatial light modulation element 90.

FIG. 5 is a schematic top plan view of the illumination region 91 whichis formed by the first spatial light modulation element 90.

FIG. 6 is a flowchart in which a genetic algorithm is used.

FIG. 7A is a schematic top plan view of the first spatial lightmodulation element 90 having the illumination region 91 with a circularshape of which the diameter is large.

FIG. 7B is a schematic top plan view of the first spatial lightmodulation element 90 having the illumination region 91 with a circularshape of which the diameter is small.

FIG. 7C is a schematic top plan view of the first spatial lightmodulation element 90 having the illumination region 91 with an annularshape.

FIG. 7D is a schematic top plan view of the first spatial lightmodulation element 90 having illumination regions 91 with four smallcircular shapes, the illumination regions 91 being axisymmetricallydisposed with respect to the optical axis.

FIG. 7E is a schematic top plan view of the first spatial lightmodulation element 90 having illumination regions 91 with twoquadrangular shapes, the illumination regions 91 being axisymmetricallydisposed with respect to the optical axis.

FIG. 7F is a schematic top plan view of the first spatial lightmodulation element 90 in which the illumination region 91 isnon-axisymmetrically formed.

FIG. 8A is a diagram illustrating examples of combinations between FIG.7A and FIG. 7B.

FIG. 8B is a diagram illustrating examples of combinations between FIG.7A and FIG. 7D.

FIG. 9A is a flowchart of method 1 of estimating phase information of aspecimen 60.

FIG. 9B is a diagram of an object information acquisition screen 23 ddisplayed on a parameter setting portion 23 of the display section 21.

FIG. 9C is a flowchart of a method of estimating microscopic structureinformation of the specimen 60.

FIG. 9D is a flowchart of a method of estimating information oncharacteristics of the wavelength of the illumination light of thespecimen 60.

FIG. 10A is a flowchart of method 2 of estimating phase information ofthe specimen 60.

FIG. 10B is a diagram of the display section 21 shown in the schematicview of the first spatial light modulation element 90.

FIG. 11 is a schematic configuration diagram of a microscope system 200.

FIG. 12 is a flowchart of a method of detecting spatial frequencyinformation of an object.

FIG. 13A is a diagram of the display section 21 which displays a pictureof an image of a pupil 273 detected by a second image sensor 280 in acase where the specimen 60 is an integrated circuit (IC).

FIG. 13B is a diagram of the display section 21 which displays a pictureof an image of the pupil 273 detected by the second image sensor 280 ina case where the specimen 60 is a biological object.

FIG. 13C is a diagram of the display section 21 which displays a pictureof an image of the pupil 273 in each wavelength of red, blue, and greendetected by the second image sensor 280 in a case where the specimen 60is a biological object.

FIG. 14A is a schematic configuration diagram of a microscope system300.

FIG. 14B is a top plan view of a first spatial light modulation element390.

FIG. 14C is a top plan view of a second spatial light modulation element396.

DESCRIPTION OF EMBODIMENTS FIRST EXAMPLE

As a first example, a description will be given of a microscope system100 which is automatically adjusted by deriving the intensitydistribution of the illumination light appropriate to observe the imageof the object in a better condition during the observation by freelychanging the shape of the diaphragm.

<Microscope System 100>

FIG. 1 is a schematic configuration diagram of the microscope system100. The microscope system 100 mainly includes: an illumination lightsource 30; an illumination optical system 40; a stage 50; an imagingoptical system 70; an image sensor 80; and a calculation section 20.Hereinafter, the center axis of rays emitted from the illumination lightsource 30 is set as the Z axis direction, and the directions, which areperpendicular to the Z axis and are orthogonal to each other, are set asthe X axis direction and the Y axis direction.

The illumination light source 30 illuminates white illumination lighton, for example, the specimen 60. The illumination optical system 40includes a first condenser lens 41, a wavelength filter 44, a firstspatial light modulation element 90, and a second condenser lens 42.Furthermore, the imaging optical system 70 includes an objective lens71. The stage 50 is movable in the XY axis directions in a state wherethe specimen 60 having an unknown structure of, for example, celltissues or the like is placed. Furthermore, the imaging optical system70 forms an image of the transmitted light or the reflected light of thespecimen 60 on the image sensor 80.

The first spatial light modulation element 90 of the illuminationoptical system 40 is disposed, for example, at a position conjugate tothe position of the pupil of the imaging optical system 70 in theillumination optical system 40, and is able to change the intensitydistribution of the illumination light at the conjugate position of thepupil of the imaging optical system 70. Furthermore, the first spatiallight modulation element 90 has an illumination region 91 of which theshape and the size are freely changeable, and is able to arbitrarilychange the intensity distribution of the illumination light by changingthe size or the shape of the illumination region 91. Furthermore, awavelength filter 44 limits the wavelength of the transmitted rayswithin a specific range. As the wavelength filter 44, for example, abandpass filter for transmitting only light with the wavelength in thespecific range is used. The wavelength filter 44 is removable, and isreplaced by providing a bandpass filter for transmitting a plurality ofrays with respective different wavelengths. Thereby, it is possible tocontrol the wavelength of the light transmitted through the wavelengthfilter 44.

The calculation section 20 receives the output data which is detected bythe image sensor 80, and displays the output data on the display section21 such as a monitor. Furthermore, by analyzing the output data, theintensity distribution of the illumination light appropriate for theobservation of the specimen 60 is calculated. Furthermore, thecalculation section 20 is able to perform control of the wavelengthrange of the rays, which are transmitted through the wavelength filter44, such as control and drive of the illumination region 91 of the firstspatial light modulation element 90.

In FIG. 1, the light, which is emitted from the illumination lightsource 30, is indicated by the dotted line. The light LW11, which isemitted from the illumination light source 30, is converted into theparallel light LW12 through the first condenser lens 41. The light LW12is transmitted through the wavelength filter 44 such that the wavelengthrange of the light LW12 is specified, and is incident to the firstspatial light modulation element 90. The light LW13, which passesthrough the illumination region 91 of the first spatial light modulationelement 90, is transmitted through the second condenser lens 42, isconverted into the light LW14, and propagates toward the stage 50. Thelight LW15, which is transmitted through the stage 50, is transmittedthrough the imaging optical system 70, is converted into the light LW16,and forms an image of the specimen 60 on the image sensor 80.

The image of the specimen 60 detected by the image sensor 80 is sent asthe output data to the calculation section 20. The calculation section20 estimates the structure of the specimen 60 on the basis of outputdata which can be obtained from the image sensor 80, a transmissionwavelength of the wavelength filter 44, and shape data of theillumination region 91 which is formed by the first spatial lightmodulation element 90, thereby calculating the illumination shapeappropriate for the observation of the specimen 60, that is, theintensity distribution of the illumination light. Then, the shape, whichis appropriate for the observation of the specimen 60 subjected to thecalculation, is transmitted to the first spatial light modulationelement 90, and the illumination region 91 is formed in an illuminationshape which is appropriate for the observation of the specimen 60.Furthermore, likewise, the wavelength of the illumination light, whichis appropriate for the observation of the specimen 60, is alsocalculated by the calculation section 20, and a bandpass filter mostappropriate for the observation of the specimen 60 is selected as thewavelength filter 44.

<Illumination Optical System 40>

As the first spatial light modulation element 90, a liquid crystal panel93, a digital micro mirror device (DMD) 94, and the like may be used.The first spatial light modulation element 90 in a case of using theabove-mentioned components will be described with reference to FIG. 2.

FIG. 2A is a schematic configuration diagram in a case where a firstspatial light modulation element 90 is the liquid crystal panel 93. Theliquid crystal panel 93 is constituted by, for example, a liquid crystalfilm 93 a, a first polarizing film 93 b, and a second polarizing film 93c. The liquid crystal film 93 a is filled with a liquid crystalmaterial, and electrodes such as thin film transistors (TFT) are formedthereon, whereby it is possible to apply a voltage to any locations ofthe liquid crystal film 93 a. The light LW11, which is emitted from theillumination light source 30, is converted into the parallel light LW12through the first condenser lens 41, and the range of the wavelengththereof is specified by the wavelength filter 44, whereby the light islimited to only the light LW12 a which is unidirectionally polarized bythe first polarizing film 93 b. The light LW12 a is converted into thelight LW12 c, which is polarized at 90 degrees by applying a voltage tothe liquid crystal film 93 a, and the light LW12 b, which is notpolarized by not applying a voltage to the liquid crystal film 93 a,through the liquid crystal film 93 a. The second polarizing film 93 c isdisposed to transmit only light, which is polarized at 90 degrees, amonglight which is transmitted through the first polarizing film 93 b.Hence, only the light LW12 c is transmitted through the secondpolarizing film 93 c, and the light LW13 is transmitted through thesecond polarizing film 93 c. In the liquid crystal panel 93, theillumination region 91 is formed in a random shape by controlling theposition of the liquid crystal film 93 a where a voltage is applied.

FIG. 2B is a schematic configuration diagram in a case where the firstspatial light modulation element 90 is the digital micro mirror device(DMD) 94. The aggregation of a plurality of small movable reflectionmirrors (not shown in the drawing) is formed on the surface of the DMD94, and each mirror is independently movable. The light LW11, which isemitted from the illumination light source 30, is converted into theparallel light LW12 through the first condenser lens 41, and the rangeof the wavelength thereof is specified by the wavelength filter 44,whereby the light is illuminated on the entire DMD 94. When the movablereflection mirrors which are disposed in the region 94 a of the DMD 94are directed to a direction in which the light LW11 is reflected by thespecimen 60, the light LW13 a is illuminated on the specimen 60. Whenthe DMD 94 is used in the first spatial light modulation element 90, byperforming control as to which positions the movable reflection mirrormoves, it is possible to illuminate the light with a random shape to thespecimen 60. This means that the illumination region 91 of the firstspatial light modulation element 90 shown in FIG. 1 is formed in therandom shape.

As the first spatial light modulation element 90, an electrochromicelement may be used. The electrochromic element is mainly formed in astructure in which the transparent electrodes such as TFTs and theelectrochromic layer are combined and laminated. In the electrochromiclayer, when a voltage is applied, an electrolytic oxidation or reductionreaction reversibly occurs in the region to which the voltage isapplied. Thus, it is possible to reversibly change a state in which thelight is transmitted, and a state in which the light is not transmitted.Hence, in the electrochromic element, the illumination region 91 isformed in a random shape by controlling the position of theelectrochromic layer where a voltage is applied. The detaileddescription of the operation and the structure of the electrochromicelement are disclosed in, for example, Japanese Unexamined PatentApplication Publication No. H8-220568.

Furthermore, as the first spatial light modulation element 90, anoptical element may be used if the optical element has a plurality ofspaces in which an electroactive material having the specific opticalcharacteristics such as the transmittance changed by application of anelectric stimulus is enclosed and in which electrodes such as TFTs areformed. The optical element has cells which are airtightly sealed andare formed in an array shape, and the electroactive material is sealedin each cell. The electrodes are formed in the respective cells, and avoltage can be independently applied to each cell. Thus, by controllingthe voltage applied to the cell, it is possible to reversibly change astate in which light is transmitted through the cell and a state inwhich light is not transmitted through the cell. In this opticalelement, by performing control as to which cells a voltage is appliedto, the illumination region 91 is formed in a random shape. The detaileddescription of the operation and the structure of this optical elementare disclosed in, for example, PCT Japanese Translation PatentPublication No. 2010-507119.

In FIG. 1, the wavelength filter 44 is disposed between the firstcondenser lens 41 and the first spatial light modulation element 90, butin order to detect the light with a specific wavelength through theimage sensor 80, the filter may be disposed at a certain positionbetween the illumination light source 30 and the image sensor 80.

Furthermore, instead of using a light source which illuminates whiteillumination light as the illumination light source 30 and thewavelength filter 44, an LED (light emitting diode) or the like may beused as the illumination light source 30. When the illumination lightsource 30 is constituted by the LED, for example, the illumination lightsource can be constituted by combination of the LEDs emitting light withrespective wavelengths of red, blue, and green. Light on/off of the LEDwith each wavelength is controlled by the calculation section 20,thereby controlling the wavelength of the light which is emitted by theillumination light source 30. In such a manner, the LED light source canbe used instead of the combination of the wavelength filter 44 and thewhite illumination light source. Furthermore, as the image sensor 80, animage pickup device having a plurality of light receiving elements, inwhich the wavelengths of the received light are different, such as CCDand CMOS may be used. In this case, for example, by extracting a signalof the light receiving element which receives light with the wavelengthof red, it is possible to obtain the light with the wavelength of redtransmitted through the specimen 60.

<<Method of Deriving Illumination Shape (Intensity Distribution of TheIllumination Light)>>

A method of calculating an illumination shape appropriate for theobservation of the specimen 60 will be hereinafter described. As thecalculation method, there are several methods such as simulatedannealing and Tabu search. Hereinafter, two methods of the hill climbingmethod (the maximum grade method) and a method using the geneticalgorithm will be described.

<Hill Climbing Method>

The hill climbing method is a method of incrementally changing theinitially set illumination shape and acquiring output data of a picturefor each change so as to thereby find a condition in which the outputdata is most approximate to the condition set by an observer. Referringto FIG. 3, the description will be given below.

FIG. 3 is a flowchart of the hill climbing method that finds anappropriate illumination shape by incrementally changing theillumination shape.

In step S101, first, the illumination region 91 of the first spatiallight modulation element 90 is set to the size and shape of an initialsetting. For example, the illumination region 91 of the initial settinghas a circular shape of which the diameter is the maximum. In thisstate, the image of the specimen 60 is detected by the image sensor 80.It is an object of the detection of the image of the specimen 60 toacquire a reference picture before adjusting the shape and size of theillumination region 91. The output data of the picture of the image ofthe specimen 60 detected by the image sensor 80 is sent to thecalculation section 20, and then the picture of the specimen 60 isdisplayed on the display section 21 such as a monitor which is connectedto the calculation section 20.

In step S102, a region setting portion 22 (refer to FIG. 4A) on thedisplay section 21 sets an observational region 24 (refer to FIG. 4A) ofthe observed image. In step S101, the picture of the specimen 60 isdisplayed in the region setting portion 22. The region setting portion22 and the observational region 24 will be described in detail withreference to FIG. 4A.

In step S103, parameters for forming the observed image of the specimen60 are set. In the parameter setting portion 23 (refer to FIG. 4A), theobserver is able to set parameters for inputting the observationcondition, which is requested and allowed by an observer, for theobserved image of the specimen 60. Hereinafter, referring to FIG. 4A, adisplay example of the display section 21 will be described.

FIG. 4A is a diagram of the region setting portion 22 and the parametersetting portion 23 of the display section 21. It is preferable that thedisplay section 21 be formed by GUI (graphical user interface) whichperforms an input through a mouse, a touch pad, and the like. Since theobserver is able to intuitively perform operation through the GUI, theGUI facilitates the operation. The display section 21 may display, forexample, the region setting portion 22 and the parameter setting portion23. The image of the specimen 60 is displayed in the region settingportion 22, and thus it is possible to set the observational regiondesired by the observer. Furthermore, in the parameter setting portion23, it is possible to input the setting of the observation conditiondesired by the observer. For example, an observation condition settingitem display screen 23 a is displayed in the parameter setting portion23. In the observation condition setting item display screen 23 a, forexample, items such as region setting, spatial frequency band setting,and wavelength band setting are displayed. When the region setting isselected, the screen of the display section 21 is changed into theregion setting screen 22 a. Furthermore, when the spatial frequency bandsetting and the wavelength band setting are selected, the screen of thedisplay section 21 is respectively changed into the spatial frequencyband setting screen 23 b and the wavelength band setting screen 23 c.

The region setting portion 22 is represented as, for example, the regionsetting screen 22 a. In the region setting portion 22, the picture ofthe specimen 60 detected in step S101 is displayed. The observer is ableto set the observational region 24 for the observed image of thespecimen 60. For example, the observer may set the entirety of thespecimen 60 as the observational region 24, or may set only a part ofthe specimen 60. Furthermore, in the region setting portion 22, two ormore observational regions 24 may be set at a time. Furthermore, theregion setting portion 22 may display the specimen 60 by non-opticallyand electrically enlarging it such that the observer easily sets theobservational region 24, or may display the entire image of the specimen60 by reducing the picture of the specimen 60. Furthermore, theobservational region 24 is set as a region in which the parameters setby the parameter setting portion 23 are reflected.

Furthermore, the observational region 24 may be automatically set by thecalculation section 20. For example, the contrast of the picture outputdata acquired in step S101 is calculated, and the region is roughlyclassified into a high contrast region and a low contrast region. Then,the low contrast region in the region setting screen 22 a of FIG. 4A isautomatically set as the observational region 24, and thus it ispossible to optimize this observation condition in the observationalregion 24. In this example, the region is roughly classified into thehigh contrast region and the low contrast region, but the invention isnot limited to the two regions. For example, the region may beclassified into three or more regions including a mid contrast region bytimely setting of a threshold value of the contrast. Furthermore, inthis example, the low contrast region is set as the observational region24, but instead of this, the high contrast region or the mid contrastregion may be set. Furthermore, the setting of the observational region24 is not limited to the methods based on contrast. For example, on thebasis of the spatial frequency and the like derived by a method ofdetecting the spatial frequency information of an object describedbelow, the observational region 24 may be automatically set.

In the spatial frequency band setting screen 23 b, the observer is ableto set the desired spatial frequency band of the specimen 60. Thesetting of the spatial frequency band may be, as shown in FIG. 4A, madein such a way that the observer inputs numerical values, or may be madesuch that the observer is able to select the desired spatial frequencyband among a plurality of options. Furthermore, in the wavelength bandsetting screen 23 c, the observer is able to set the wavelength band ofthe light which is intended to be used or intended to be observed. Forexample, when the wavelength appropriate for the observation of thespecimen 60 is estimated in method 1 of estimating the objectinformation to be described later, the wavelength can be set by thewavelength band setting screen 23 c. The setting of the wavelength bandmay be made, as shown in FIG. 4A, in such a way that the observer inputsnumerical values, or may be made such that the observer is able toselect the desired wavelength band among a plurality of options of, forexample, red, green, and blue.

In addition, when the observer does not want to set the observationalregion 24, step S102 may be skipped. In this case, the entire image ofthe specimen 60 detected by the image sensor 80 is set as theobservational region 24.

As shown in FIG. 4A, the observer sets the parameters by using theparameter setting portion 23 of the display section 21 at the conditionwhich is requested or allowed by the observer. The examples of the setparameters include a specific location of the specimen 60 which isintended to be observed at a high contrast, a specific spatial frequencyregion of the specimen 60, and the like. For example, there may be arequest to observe the observational region 24, which is set in stepS102, by giving a shading effect thereto or to clearly observe thedetailed image of the observational region 24. In this case, theobserver is able to set the wavelength band and the spatial frequencyband.

Furthermore, the observer may initialize the illumination region 91 as asingle parameter. For example, it is possible to initialize the shape ofthe illumination region 91 which is used first in step S104 to bedescribed later. Furthermore, in step S101, in such a case where theshape of the illumination region 91 preferred for the specimen 60 can beanticipated, the shape of the illumination region 91 may be used as aninitial setting. By initializing the shape of the illumination region91, it is possible to reduce the time to finally determine the shape ofthe illumination region 91. Referring to FIG. 4B, a description will begiven of an example in the case where the observer initializes theillumination region 91.

FIG. 4B is a diagram of the display section 21 in the case ofinitializing the illumination region 91 of the first spatial lightmodulation element 90. In FIG. 4B, the parameter setting portions 23 areformed at two locations on the right and left sides of the displaysection 21. In the right-side parameter setting portion 23, the top planview of the first spatial light modulation element 90 is indicated bythe dotted line. Furthermore, in FIG. 4B, a light blocking section 92and the illumination region 91 which is formed in the light blockingsection 92 are indicated by the hatched line. Furthermore, in FIG. 4B,the coordinate lines 97 are represented such that the center axis of thefirst spatial light modulation element 90 is recognizable. The observeris able to freely initialize the shape of the illumination region 91 ofthe first spatial light modulation element 90. In order to form theillumination region 91, for example, the shape samples of theillumination region 91 may be displayed in the left-side parametersetting portion 23 of FIG. 4B, and a desired illumination region 91 maybe selected therefrom. In addition, the desired illumination region 91may be formed by freely drawing the shape thereof. Furthermore, it isnot necessary for the illumination region 91 to be disposed on thecenter axis of the first spatial light modulation element 90. That is,the observer may set, as shown in FIG. 4B, the illumination region 91 ata position far from the center axis. Furthermore, two or moreillumination regions 91 may be set at the same time.

The screen shown in FIG. 4A or 4B is selectively displayed as a windowon the display section 21. Furthermore, the display section 21 maydisplay only the region setting portion 22 or the parameter settingportion 23.

Returning to FIG. 3, in step S104, the calculation section 20 changesthe size of the illumination region 91 of the first spatial lightmodulation element 90. When the observer sets the illumination region 91in step S103, the calculation section 20 uses the set illuminationregion 91 as the initial setting value, and changes the size of theillumination region 91. When the illumination region 91 is not set instep S103, the calculation section 20 slightly changes the size of theillumination region 91 of the initial setting value which is set in stepS101. That is, the intensity distribution of the illumination light isslightly changed.

Referring to FIG. 5, the change of the intensity distribution of theillumination light will be described. FIG. 5 is a schematic top planview of the first spatial light modulation element 90. In FIG. 5, thecircular illumination region 91 is formed on the center portion of thelight blocking section 92 of the first spatial light modulation element90. FIG. 5 shows an example in the case where the illumination region 91is initialized as a circle of which the diameter is W13 at the center ofthe first spatial light modulation element 90 in step S103.

The diameter of the light blocking section 92 is W11, and the diameterof the illumination region 91 of the initial setting is W13. Then, thesize of the illumination region 91 is slightly changed in step S104, andthe diameter of the illumination region 91 is changed to W12. In theexample of FIG. 5, the calculation section 20 changes the diameter ofthe illumination region 91 from the diameter W13 to the diameter W12which is slightly larger than W13, where the change is performed suchthat the intensity distributions of the illumination light thereof aresimilar to each other.

In step S105, the image sensor 80 detects the image of the specimen 60.For example, in FIG. 5, under the condition of the illumination region91 of which the diameter is changed to the diameter W12, the image ofthe specimen 60 is detected by the image sensor 80, and the output datais sent to the calculation section 20.

In step S106, it is determined whether or not the current output data,which is sent to the calculation section 20, is worse than the previousoutput data. For example, the observational region 24 is set by theregion setting portion 22 of the display section 21 shown in FIG. 4A,and it is assumed that the setting where the contrast of theobservational region 24 is intended to increase is performed by theparameter setting portion 23. A comparison is performed as to whether ornot the contrast, which is calculated on the basis of the currentlyobtained output data (for example, when the illumination region 91 hasthe diameter W12), is worse than the contrast which is calculated on thebasis of the previously obtained output data (for example, when theillumination region 91 has the diameter W13). If it is not worse, theprocedure returns to step S104, the diameter of the illumination region91 is changed, and the output data is detected (step S105). That is,since the contrast of the observational region 24 increases, theprocedure returns to step S104, and the size of the illumination region91 is further changed. In contrast, if the current contrast is worsethan the previous contrast, the diameter of the previous illuminationregion 91 has the maximum contrast. Accordingly, the procedure advancesto the next step S107.

In step S107, the illumination shape, which is appropriate for theobservation of the specimen 60, is selected. That is, the illuminationshape, which is used just before the contrast of the observationalregion 24 gets worse, is assumed as an illumination shape for theobservation of the specimen 60, and is used in the observation of thespecimen 60.

In step S104 of the flowchart, the size of the illumination region 91 ischanged in a similar shape. However, not only the change into a similarshape, but also a change of the shape itself may be performed. Forexample, the circular illumination region 91 may be incrementally shapedto be finally formed in a triangular shape, or the circular illuminationregion 91 may be incrementally shaped to be finally formed in an annularshape with a predetermined width.

<Method Using Genetic Algorithm>

Next, a method using the genetic algorithm will be described. Thegenetic algorithm is a method of finding an illumination shape byacquiring the picture data pieces, which are respectively associatedwith a plurality of illumination shapes provided in advance, andperforming combination of the illumination shapes appropriate for theobservation of the specimen 60.

FIG. 6 is a flowchart in which the genetic algorithm is used.

First, in step S201, the illumination region 91 of the first spatiallight modulation element 90 is set to have the size and the shape of theinitial setting. For example, the illumination region 91 of the initialsetting has a circular shape with the maximum diameter. In this state,the image of the specimen 60 is detected by the image sensor 80.

In step S202, the observational region 24 of the observed image is setby the region setting portion 22. Through step S201, the picture of theimage of the specimen 60 is displayed in the region setting portion 22.

In step S203, parameters for forming the observed image of the specimen60 are set. In the parameter setting portion 23, the observer is able toset parameters for inputting the observation condition, which isrequested and allowed by the observer, for the observed image of thespecimen 60. Like parameters shown in FIG. 4A, examples of the setparameters include the specific location of the specimen 60, the spatialfrequency band and the wavelength band of the specimen 60, and the like.However, as shown in FIG. 4B, it is not necessary for the observer toset the illumination region 91 of the first spatial light modulationelement 90. The calculation section 20 arbitrarily initializes theillumination region 91.

In step S204, by using two or more initial plural illumination shapes,the images of the specimen 60 are detected by the image sensor 80. Then,the calculation section 20 acquires the respective pieces of the outputdata of the pictures of the image by which the specimen 60 is measuredby using the plural illumination shapes. Referring to FIG. 7, examplesof the plural illumination shapes will be described.

FIG. 7 shows diagrams of various illumination shapes of the firstspatial light modulation element 90. In FIG. 7, the outlined partrepresents the illumination region 91, and the hatched region representsthe light blocking section 92.

FIG. 7A is a schematic top plan view of the first spatial lightmodulation element 90 having the circular illumination shape of whichthe diameter is large. The illumination shape of the first spatial lightmodulation element 90 may be, as shown in FIG. 7A, a circular shapewhich is axisymmetric with respect to the optical axis of theillumination optical system. FIG. 7B is a schematic top plan view of thefirst spatial light modulation element 90 having the circularillumination shape of which the diameter is small. FIG. 7B shows acircular shape in which only the size of the illumination shape isdifferent from that of FIG. 7A and which is axisymmetric with respect tothe optical axis of the illumination optical system 40. The illuminationshape of the first spatial light modulation element 90 may include, asshown in FIG. 7B, a figure with a shape similar to the other figure.FIG. 7C is a schematic top plan view of the first spatial lightmodulation element 90 having the large annular illumination shape. InFIG. 7C, light is blocked at the center portion of the large circularillumination shape of FIG. 7A.

Furthermore, FIG. 7D is a schematic top plan view of the first spatiallight modulation element 90 having the illumination shape in which theillumination regions 91 with four circular shapes each having a smalldiameter are axisymmetrically disposed with respect to the optical axis.FIG. 7E is a schematic top plan view of the first spatial lightmodulation element 90 having the illumination shape in which theillumination regions 91 with two quadrangular shapes are provided andare axisymmetrically disposed with respect to the optical axis.

FIG. 7F is a schematic top plan view of the first spatial lightmodulation element 90 in which the illumination region 91 isnon-axisymmetrically formed with respect to the optical axis. In FIG.7F, the illumination shape of the first spatial light modulation element90 is formed as a crescent shape, and is non-axisymmetric with respectto the optical axis. Normally, the non-axisymmetric illumination region91 is mostly used in inclined illumination, and thus it is possible toincrease the contrast of the specimen 60. However, the non-axisymmetricillumination region 91 increases the contrast of only a part of theobject, whereby a picture having an uneven contrast is obtained, andthus is inappropriate to observe the entire specimen 60. Hence, for thegenetic algorithm, it is preferable that the parameter setting portion23 of the display section 21 shown in FIG. 4A be configured such thatwhether or not to use the non-axisymmetric opening is selectable.

Returning to FIG. 6, in step S205, by comparing the respective pieces ofthe output data of the picture of the specimen 60 acquired in step S204,the first intensity distribution of the illumination light, which isformed in an illumination shape most appropriate for the set parametersamong the output data pieces, and the second intensity distribution ofthe illumination light, which is formed in an illumination shapesecondarily appropriate therefor, are selected.

In step S206, the calculation section 20 forms illumination shapes,which have next-generation intensity distributions of the illuminationlight, from the first illumination intensity distribution and the secondillumination intensity distribution in accordance with a method ofcrossover or mutation of the genetic algorithm. Referring to FIG. 8, adescription will be given of an example in which the illumination shapeshaving the next-generation intensity distributions of the illuminationlight are formed.

FIG. 8A is a diagram illustrating examples of combinations of the firstspatial light modulation element 90 between FIG. 7A and FIG. 7B. In stepS205, the first intensity distribution of the illumination light has acircular illumination shape with a large diameter shown in FIG. 7A, andthe second intensity distribution of the illumination light has acircular illumination shape with a small diameter shown in FIG. 7B. Thecalculation section 20 is able to form a plurality of new illuminationshapes by crossing (combining) the two shapes. Examples of the formedillumination shape include: a first spatial light modulation element 90a that has an illumination region 91 with a diameter which is slightlysmaller than that of the illumination region 91 of FIG. 7A; a firstspatial light modulation element 90 b that has an illumination region 91with a diameter which is slightly larger than that of the illuminationregion 91 of FIG. 7B; a first spatial light modulation element 90 c inwhich the illumination region 91 is formed in an ellipse shape; and thelike.

FIG. 8B is a diagram illustrating examples of combinations of the firstspatial light modulation element 90 between FIG. 7A and FIG. 7B. In stepS205, the first intensity distribution of the illumination light has acircular illumination shape with a large diameter shown in FIG. 7A, andthe second intensity distribution of the illumination light has anillumination shape in which the four small circular illumination regions91 shown in FIG. 7D are axisymmetrically disposed with respect to theoptical axis. By crossing (combining) the two shapes, the calculationsection 20 is able to form, for example: a first spatial lightmodulation element 90 d that has illumination regions 91 having a shapein which four parts of a circular ring are axisymmetrically disposedwith respect to the optical axis; a first spatial light modulationelement 90 e in which an illumination region 91 is formed in the shapeof “X”; and the like.

FIGS. 8A and 8B are just examples of the combinations. In practice, theshape of the first spatial light modulation element 90 is randomlyformed, and thus shapes of the illumination regions 91 newly formed areinnumerable. The number of the shapes of the illumination regions 91 maybe a few. Furthermore, the combination may be performed by using adifferent method. For example, the first spatial light modulationelement 90 may be divided into a plurality of microscopic regions, andoperations such as recombination and mutation may be performed on eachregion. Furthermore, by creating an independent function, thecombination may be performed in terms of the function.

Returning to FIG. 6, in step S207, the first illumination intensitydistribution, which is most appropriate for the observation of thespecimen 60, and the second illumination intensity distribution, whichis secondarily appropriate therefor, are selected from the firstillumination intensity distribution, the second illumination intensitydistribution, and the next-generation illumination intensitydistributions.

In step S208, it is determined whether crossover or mutation isperformed up to a predetermined generation, for example, 1000generations. If crossover or the like is not performed up to thepredetermined generation, the procedure returns to step S206, and theillumination intensity distribution further appropriate for theobservation of the specimen is searched. If crossover or the like isperformed up to the predetermined generation, the procedure advances tostep S209.

In step S209, from the illumination regions 91 which are obtained bycrossover or the like up to the predetermined generation, for example,the 1000 generations, the illumination shapes at a generationapproximate to the condition requested by the observer is selected.Thereafter, the first spatial light modulation element 90 of theillumination shape at the generation is used in the observation of thespecimen 60.

<<Method 1 of Estimating Object Information>>

When the structure or the characteristic of the specimen 60 is unknown,it is preferable that structure or characteristic information of thespecimen 60 be acquired before the illumination shape, which is mostappropriate to the specimen 60, is derived. The reason is that, byreferring to the structure or the characteristic of the specimen 60 inthe case of estimating the most appropriate observation condition, it ispossible to reliably obtain the most appropriate observation conditionfor a shorter period of time. Hereinafter, a description will be givenof the method of estimating phase information of the specimen 60,microscopic structure information, and information on characteristics ofthe wavelength of the illumination light.

<Estimation Method 1 of Phase Information of Object>

By changing the shape of the illumination region 91 of the first spatiallight modulation element 90 and observing the specimen 60, it ispossible to estimate whether the contrast of the specimen 60 is anintensity object of which the contrast is high or a phase object ofwhich the contrast is low. Whether or not the specimen 60 is the phaseobject or the intensity object can be estimated by illuminating rayswith different values of the coherence factor (σ) on the specimen 60.The value of σ is defined by σ=NA′/NA. NA′ is a numerical aperture ofthe illumination optical system 40, and NA is a numerical aperture ofthe objective lens 71. The numerical aperture NA′ of the illuminationoptical system 40 can be controlled by changing the shape of theillumination region 91 of the first spatial light modulation element 90.In NA′, the illumination region 91 is assumed as a point shape(hereinafter referred to as a point light source), whereby the value ofσ is regarded as 0. Furthermore, when the illumination region 91 of thefirst spatial light modulation element 90 has a circular shape of whichthe diameter is large, NA′ is equal to 1.

FIG. 9A is a flowchart of method 1 of estimating phase information of aspecimen 60.

First, in step S301, the observer selects the phase information of theobject information acquisition screen 23 e displayed on the displaysection 21 of FIG. 9B.

FIG. 9B is a diagram of the region setting portion 22 and the parametersetting portion 23 of the display section 21 in the method of estimatingobject information. First, an object information acquisition screen 23 dis displayed on the parameter setting portion 23 of the display section21. The observer selects the object information detection 1 whenperforming method 1 of estimating the phase information of the objectthrough the object information acquisition screen 23 d, selects theobject information detection 2 when performing method 2 of estimatingthe phase information of the object to be described later, and selectsbatch measurement when performing both of the method 1 of estimating thephase information of the object and the method 2 of estimating the phaseinformation of the object. When method 1 of estimating the phaseinformation of the object is selected, the screen is changed into theobject information acquisition screen 23 e, and when method 2 ofestimating the phase information of the object is selected, the screenis changed into the object information acquisition screen 23 f.Furthermore, the object information acquisition screen 23 g is a screenwhich is changed from the object information acquisition screen 23 e.

The object information acquisition screen 23 e displays items of thephase information 1, the microscopic structure, characteristics of thewavelength, and the batch measurement. Here, when the phase information1 is selected, the calculation section 20 performs method 1 ofestimating the phase information of the object. When the microscopicstructure is selected, the calculation section 20 performs the method ofestimating information of the microscopic structure of the object. Whenthe characteristics of the wavelength are selected, the calculationsection 20 performs the method of estimating the information of thecharacteristics of the wavelength of the object. Furthermore, when thebatch measurement is selected, the calculation section 20 performs allestimations of the items. After each selection item is selected,information of the selected item is automatically acquired.

Returning to FIG. 9A, in step S302, the shape of the illumination region91 of the first spatial light modulation element 90 is formed as a pointlight source (σ≈0), and the image of the specimen 60 formed by theillumination of the point light source is detected by the image sensor80. If the irradiated light is coherent, the contrast is observed in thespecimen 60 even when the specimen 60 is the phase object or theintensity object.

Next, in step S303, the shape of the illumination region 91 of the firstspatial light modulation element 90 is formed as a circular shape (σ≈1)of which the diameter is large, and the image of the specimen 60 formedby the illumination of the large circular shape is detected by the imagesensor 80. If the irradiated light is incoherent, the specimen 60 as theintensity object can be observed since the contrast is present in thespecimen 60, but the specimen 60 as the phase object cannot be observedsince the contrast is absent in the specimen 60.

Subsequently, in step S304, it is estimated whether the specimen 60 isthe phase object or the intensity object. If there is no change betweenthe image formed by the coherent light detected in step S302 and theimage formed by the incoherent light detected in step S303, it isestimated that the specimen 60 is the intensity object. If there is adifference between the image formed by the coherent light detected instep S302 and the image formed by the incoherent light detected in stepS303, it is estimated that the specimen 60 is the phase object.

Next, in step S305, the shape of the illumination region 91 appropriatefor the observation of the specimen 60 is estimated. As in the phaseobject, the calculation section 20 sets the shape of the illuminationregion 91 of the first spatial light modulation element 90 as small orinclined illumination (for example, refer to FIG. 7F). The reason isthat, when the specimen 60 is the phase object, small or inclinedillumination in the shape of the illumination region 91 is appropriatefor the observation of the specimen 60. Furthermore, when the specimen60 is the intensity object, the calculation section 20 increases thediameter of the circle of the illumination region 91 of the firstspatial light modulation element 90. The reason is that the intensityobject can be easily observed when the amount of light is great.

<Estimation Method of Microscopic Structure Information of Object>

Whether or not a microscopic structure is included in the specimen 60can be estimated by changing the shape of the illumination region 91 ofthe first spatial light modulation element 90 and observing the specimen60.

FIG. 9C is a flowchart of a method of estimating the microscopicstructure information of the specimen 60. First, in step S311, theobserver selects the microscopic structure information on the objectinformation acquisition screen 23 e displayed on the display section 21of FIG. 9B.

Next, in step S312, the shape of the illumination region 91 of the firstspatial light modulation element 90 is formed as a point light source,and the image of the specimen 60 is detected by the image sensor 80.When the shape of the illumination region 91 of the first spatial lightmodulation element 90 is the point light source (σ=0), even if thespecimen 60 includes a microscopic structure, the microscopic structuredoes not appear in the image of the specimen 60.

Subsequently, in step S313, the shape of the illumination region 91 ofthe first spatial light modulation element 90 is formed as an annularshape, the image of the specimen 60 is detected by the image sensor 80.At this time, it is preferable that the contour of the annular shape belarge. When the shape of the illumination region 91 is the annularshape, if the specimen 60 includes a microscopic structure, themicroscopic structure is detected.

Next, in step S314, it is estimated whether or not the specimen 60includes a microscopic structure. If there is no change between theimages of the specimen 60 obtained when the illumination region 91 isformed as the point light source and when it is formed as the annularshape, the calculation section 20 determines that the specimen 60 doesnot include a microscopic structure. In contrast, there may be adifference in the output data between the images of the specimen 60obtained when the illumination region 91 is formed as the point lightsource and when it is formed as the annular shape, and the image of thespecimen 60 may be detected when the illumination region 91 is formed inthe annular shape. In this case, the calculation section 20 determinesthat the specimen 60 includes a microscopic structure.

Thereafter, in step S315, the shape of the illumination region 91appropriate for the observation of the specimen 60 is estimated. Forexample, if the specimen 60 includes a microscopic structure, it ispreferable that the illumination region 91 be formed in the annularshape or the like.

<Method of Estimating Information on Characteristics of Wavelength ofIllumination Light of Object>

When the wavelength of the illumination light on the specimen 60 ischanged, different output data may be shown due to the structure and thecharacteristics of the specimen 60. Hence, it is preferable to grasp thecharacteristics of the wavelength of the illumination light of thespecimen 60.

FIG. 9D is a flowchart of a method of estimating information oncharacteristics of the wavelength of the illumination light of thespecimen 60.

First, in step S321, the observer selects the information on thecharacteristics of the wavelength on the object information acquisitionscreen 23 e displayed on the display section 21 of FIG. 9B.

Next, in step S322, the illumination light illuminated on the specimen60 is generated as monochromatic light, and the image of the specimen 60is detected by the image sensor 80. For example, it is assumed that theillumination light source 30 employs LEDs having light sources of threecolors of red, blue, and green. In this case, for example, only thegreen LED is turned on, and the LEDs with different wavelengths areturned off. Then, the image of the specimen 60 is detected by the imagesensor 80.

Subsequently, in step S323, it is determined whether the images of thespecimen 60 are detected at all wavelengths. For example, if the imageof the specimen 60 is detected at each wavelength of red, blue, andgreen, the procedure advances to step S325. If there is a wavelength atwhich the image of the specimen 60 is not yet detected, the procedureadvances to step S324.

Next, in step S324, as the wavelength of the illumination light source30, the wavelength at which the image of the specimen 60 is not yetacquired is selected, and the image of the specimen 60 is detected bythe image sensor 80. For example, if only the image of the specimen 60at the wavelength of green is acquired, the red or blue LED is turnedon, and the LEDs with different wavelengths are turned off. In thisstate, the image of the specimen 60 is detected by the image sensor 80.Thereafter, the procedure returns to step S323 again, and it is verifiedwhether or not the images of the specimen 60 are detected at all thewavelengths.

In step S325, the characteristics of the wavelength of the illuminationlight of the specimen 60 are estimated. The images of the specimen 60detected in steps S322 and S324 are compared. For example, if the imageof the specimen 60 detected at the wavelength of blue is better incontrast than the images of the specimen 60 at different wavelengths,the calculation section 20 determines that the specimen 60 has finecontrast at the wavelength of blue.

Next, in step S326, the calculation section 20 estimates theillumination light with the wavelength most appropriate for theobservation of the specimen 60. For example, when the specimen 60 isintended to be observed with the maximum contrast given thereto, andwhen the wavelength of blue is used in step S325, the image of thespecimen 60 may be observed with the contrast, which is greater thanthat of the images detected at different wavelengths, given thereto. Inthis case, the calculation section 20 determines that the illuminationlight with the wavelength of blue is appropriate for the observation ofthe specimen 60.

In this method of estimating the information on characteristics of thewavelength of the illumination light of the object, the shape of theillumination region 91 may be a random shape. However, by using themethod together with the above-mentioned method 1 of estimating thephase information of the object and the method of estimating themicroscopic structure information of the object, sometimes, it may bepossible to further reliably estimate the phase information and themicroscopic structure information of the object. In this case, after thephase information or the microscopic structure information is selectedthrough the object information acquisition screen 23 e shown in FIG. 9B,by changing the screen into the object information acquisition screen 23g shown in FIG. 9B, whether or not to change the wavelength may beselectable.

By performed the above-mentioned method 1 of estimating the objectinformation before performing the above-mentioned method of deriving theillumination shape, it is possible to reduce the time to derive theillumination shape.

<<Method 2 of Estimating Object Information>>

In the flowchart shown in FIGS. 3 and 6, in steps S103 and S203, theparameters for setting the observational image of the specimen 60 areset. As in the line width of the integrated circuit of the semiconductoror the like, when it is possible to obtain the object information of thespecimen 60 in advance, the observer is able to set the parameters onthe basis of the information. However, when the specimen 60 is abiological object, in most cases, the object information of the specimen60 may not be obtained, and thus the observer may not know which thebetter way to set parameters is. Furthermore, the information, which isobtained by method 1 of estimating the object information, may beinsufficient. In such a case, before the illumination shape isdetermined, the object information may be further specifically checkedout. Hereinafter, method 2 of estimating the phase information of theobject and the method of detecting the spatial frequency information ofthe object will be described.

Method 2 of estimating the object information is performed in such a waythat the observer selects the object information detection 2 through theobject information acquisition screen 23 d of FIG. 9B. After the objectinformation detection 2 is selected, the screen is changed into theobject information acquisition screen 23 f. The object informationacquisition screen 23 f displays items of the phase information 2, thespatial frequency information, and the batch measurement. Here, when thephase information 2 is selected, method 2 of estimating the phaseinformation of the object is performed, and when the spatial frequencyinformation is selected, the method of detecting the spatial frequencyinformation of the object is performed. Furthermore, when the batchmeasurement is selected, all estimations of the items are performed.After each selection item is selected, information of the selected itemis automatically acquired.

<Method 2 of Estimating Phase Information of Object>

The phase information of the object can be estimated by measuring thespecimen 60 in a state where the illumination region 91 of the firstspatial light modulation element 90 has a minimum size so as to beformed as the point light source and the illumination light is set asmonochromatic light. Regarding the wavelength of the monochromaticlight, when the wavelength appropriate for the observation of thespecimen 60 is estimated through method 1 of estimating the objectinformation or the like, it is preferable that the wavelength be set asa wavelength of the illumination light.

Method 2 of estimating the phase information of the object is performedby selecting the phase information 2 through the object informationacquisition screen 23 f of FIG. 9B. Hereinafter, referring to FIG. 10A,method 2 of estimating the phase information of the specimen 60 will bedescribed.

FIG. 10A is a flowchart of method 2 of estimating phase information ofthe specimen 60.

First, in step S401, the observer selects the phase information 2through the object information acquisition screen 23 f of FIG. 9B.Thereafter, the display section 21 is changed to the screen shown inFIG. 10B to be described later.

First, in step S402, the observer designates the number of the pointlight sources and the formation positions of the respective point lightsources through the screen displayed on the display section 21 of FIG.10B. Hereinafter, referring to FIG. 10B, examples of the number of thepoint light sources and the formation positions of the respective pointlight sources will be described.

FIG. 10B is a diagram of the display section 21 shown in the schematicview of the first spatial light modulation element 90. In FIG. 10B, acircle 31, which is indicated by the dotted line, in the region settingportion 22 of the display section 21 represents a state in which thediameter of the light transmitted through the first spatial lightmodulation element 90 is maximized. The point light source (σ≅0), whichis used in the estimation of the phase information of the specimen 60,is shaped into a point light source or is shaped to have a size regardedas a point light source. That is, the calculation section 20 forms theillumination regions 91 having point shapes in the first spatial lightmodulation element 90 inside the circle 31 which is indicated by thedotted line. In FIG. 10B, the number of the point light sources measuredby the parameter setting portion 23 of the display section 21 and thewavelength of the used light can be set. When the number of the pointlight sources is set to 5, a total of 5 point light sources are formedat, for example, the center point (on the optical axis) of the circle 31and points having positive and negative maximum values on the X and Yaxes. Furthermore, the wavelength of the light can be input. Regardingthe wavelength, only one wavelength may be input, and a plurality ofwavelengths may be input. In this case, measurement of 5 point lightsources is performed on each wavelength.

The point light source at the center point of the circle 31 isrepresented by the black point of the point light source 32 a, the pointlight sources having the positive and negative maximum values on the Xaxis are respectively represented by the black points of the point lightsources 32 b and 32 d, and the point light sources having the positiveand negative maximum values on the Y axis are respectively representedby the black points of the point light sources 32 c and 32 e. It ispreferable that the point light source include a point light sourcewhich is formed in the vicinity of the outermost peripheral portion ofthe circle 31. The reason is that the coherent light can be incident tothe specimen 60 at various angles and thus it is possible to obtaindiffracted light at the time of the inclined illumination.

In addition, the observer does not set the number of the point lightsources and the positions of the respective point light sources, but thecalculation section 20 may automatically set the number of the pointlight sources shown in FIG. 10B to 5.

Next, in step S403, the wavelength of the illumination light is set tobe monochromatic, and the first spatial light modulation element 90forms the point light sources at predetermined positions. Themonochromatic illumination light is formed through the wavelength filter44 which transmits only light with a specific wavelength in, forexample, the white illumination light source 30. Furthermore, the firstspatial light modulation element 90 forms the illumination region 91having a size of a single point light source shown in FIG. 10B. In astate of the point light source and in a state where the light with amonochromatic wavelength are coherent, its coherency increases. Hence,this is advantageous in estimating the phase information of the specimen60.

In step S404, the image of the specimen 60 formed by the point lightsource and the light with a monochromatic wavelength is detected by theimage sensor 80.

In step S405, the calculation section 20 determines whether or not thepictures formed by the light with a monochromatic wavelength and at thepositions of all the point light sources are acquired. For example, ifall the 5 point light sources shown in FIG. 10B are not measured, theprocedure advances to step S406. If all the designated point lightsources are measured, the procedure advances to step S407.

In step S406, the calculation section 20 is able to change the positionsof the illumination regions 91 serving as the point light sources. Forexample, in step S404, the point light source 32 a of FIG. 10B may bemeasured, and the point light source 32 b may not be measured. In thiscase, the position of the illumination region 91 is formed only at theposition of the point light source 32 b. Thereafter, the procedureadvances to step S404.

In step S407, the calculation section 20 analyzes the diffracted lightof the specimen 60. For example, from the analyzed diffracted lightinformation, it is possible to find distribution of the diffracted lighthaving a specific spatial frequency component in the observationalregion 24, and thus it is possible to efficiently find the illuminationshape appropriate for the observation.

In step S408, the calculation section 20 sets an appropriateillumination condition. Furthermore, the analyzed diffracted lightinformation is displayed on the display section 21, and the observerestimates the phase information of the specimen 60 by referring to thediffracted light information. The observer is able to set theobservational region 24 and the parameters on the basis of the estimatedphase information through steps S102 and S103 of FIG. 3 and steps S202and S203 of FIG. 6.

In the above-mentioned method 2 of estimating the phase information ofthe specimen 60, measurement may be performed for each of the pluralityof wavelengths. Hence, in the flowchart shown in FIG. 10A, there may beprovided a step for checking whether or not all the wavelengths set inFIG. 10B are measured.

<Method of Detecting Spatial Frequency Information of Object>

The spatial frequency represents the cycle of the iteration of the unitlength of the specimen 60. That is, similar structures are highly likelyto be collected in a location in which the same spatial frequencies areconcentrated. Hence, the information on the spatial frequency of thespecimen 60 is used as a reference of the setting of the observationalregion 24 and the parameters in steps S102 and S103 of FIG. 3 and stepsS202 and S203 of FIG. 6. The spatial frequency information of thespecimen 60 is detected by acquiring the output data of the picture ofthe pupil of the imaging optical system 70. Furthermore, through method2 of estimating the phase information of the specimen 60, the specimen60 is measured by using the above-mentioned point light source of themonochromatic wavelength. Hereinafter, referring to FIGS. 11 and 12, themethod of detecting the spatial frequency information of the specimen 60will be described.

FIG. 11 is a schematic configuration diagram of a microscope system 200.Hereinafter, the elements common to those of the microscope system 100described in FIG. 1 will be represented by the same reference numerals,and a detailed description thereof will be omitted.

In the microscope system 200, a beam splitter 272 is disposed at theposition of the pupil 273 of the imaging optical system 70 or in thevicinity thereof. Furthermore, the microscope system 200 has a relaylens 243 which relays the split light LW21 and a second image sensor 280which is disposed at a position conjugate to the position of the pupil273. The beam splitter 272 splits light from the imaging optical system70. The split light LW21 is incident to the second image sensor 280through the relay lens 243. The light LW21 is transmitted through therelay lens 243, and is converted into the light LW22, and the light LW22forms an image of the pupil 273 on the second image sensor 280. Theinformation of the image of the pupil 273 formed on the second imagesensor 280 is sent to and analyzed by the calculation section 20.

FIG. 12 is a flowchart of a method of detecting spatial frequencyinformation of the specimen 60.

First, in step S501, the observer selects the spatial frequencyinformation through the object information acquisition screen 23 f ofFIG. 9B. Thereafter, the display section 21 is changed to the screenshown in FIG. 10B.

Next, in step S502, the observer designates the number of the pointlight sources (σ=0) and the formation positions of the respective pointlight sources through the display section 21.

Next, in step S503, the wavelength of the illumination light is set tobe monochromatic, and an opening with a size close to that of the pointlight source at a predetermined position is formed.

Steps S502 and S503 are the same as steps S402 and S403 of FIG. 10A.Furthermore, a description will be given of an example in which thenumber of the formed point light sources is 5 similarly to FIG. 10B.

In step S504, the image of the specimen 60 in the pupil 273 is detectedby the second image sensor 280. For example, if the point light source32 a shown in FIG. 10B is designated in step S503, by using only thepoint light source 32 a as the illumination light source, the picture ofthe specimen 60 is detected by the second image sensor 280.

FIG. 13A is a diagram of the display section 21 which displays thepicture of the image of the pupil 273 detected by the second imagesensor 280 in a case where the specimen 60 is an integrated circuit(IC). The picture is displayed on, for example, the region settingportion 22 of the display section 21. The circle 233, which is indicatedby the dotted line of FIG. 13A, is defined as a range capable of passingthe light that can be transmitted. The picture data, which is detectedby the second image sensor 280, is light intensity distribution in thepupil 273. The light intensity distribution is, for example, representedby the points 234 of FIG. 13. The position of the point 234 is adetection position of the signal, and the size reflects the size of thepoint light source. In FIG. 13A, the black point 234 represents that thedetected signal is strong, the white point 234 represents that thedetected signal is weak, the gray point 234 represents that the detectedsignal has an intermediate intensity between the black point 234 and thewhite point 234. The points 234 are actually displayed in a reducedmanner, and have almost no size. However, in FIG. 13A, for convenienceof description, the points 234 have sizes, and the points 234 havedifferent colors in order to represent the intensities of the signals.In FIG. 13A, the black points 234 are collected in the upper rightregion of the picture, and the white points 234 are collected in thelower left portion of the screen. This means that the spatial frequencyin the upper right portion of the screen is large and the spatialfrequency in the lower left portion of the screen is small. Furthermore,the IC has a periodic structure, and thus the points 234 detected by thesecond image sensor 280 tend to be periodically detected.

FIG. 13B is a diagram of the display section 21 which displays thepicture of the image of the pupil 273 detected by the second imagesensor 280 in a case where the specimen 60 is a biological object. InFIG. 13B, the points 234, which are detected by the second image sensor280, are displayed on the display section 21. In FIG. 13B, the points234 are represented by points having no size. The points 234 shown inFIG. 13B have signals with different intensities at the respectivepoints similarly to FIG. 13A. When the specimen 60 is a biologicalobject, as indicated by the points 234 of FIG. 13B, the points 234 haveno periodicity, and most of them are randomly shown. The reason is thatthe number of the periodic structures of the biological object issmaller than that of the IC having periodic structures shown in FIG.13A.

In step S505, it is determined whether or not the light intensityinformation at the positions of all the point light sources, forexample, at the five points is acquired. If the light intensityinformation of all the point light sources is not acquired, theprocedure advances to step S506. If the light intensity information ofall the point light sources is acquired, the procedure advances to stepS507.

In step S506, the positions of the illumination regions 91 serving asthe point light sources are changed. For example, the point light source32 a of FIG. 10B may be measured in step S504, and the point lightsource 32 b may not be measured. In this case, the illumination region91 is formed only at the position of the point light source 32 b.Thereafter, the procedure advances to step S504.

In step S507, the Fourier spectrum of the specimen 60 is measured,thereby calculating the spatial frequency distribution of the specimen60. The spatial frequency distribution may be, as shown in FIG. 13,represented as the light intensity distribution, and the light intensitydistribution is converted into the spatial frequency and is displayed onthe display section 21. From the spatial frequency distribution of thespecimen 60, the periodicity of the structure of the specimen 60 iscalculated. As shown in FIG. 13B, if the spatial frequency distributionof the specimen 60 is random, in this state, the periodicity of thestructure of the specimen 60 cannot be calculated.

In step S508, the calculation section 20 sets the illumination conditionappropriate for the observation of the specimen 60. Furthermore, theresult thereof may be displayed on the display section 21. The observersets the parameters or sets the observational region 24 through thedisplay section 21 on the basis of the spatial frequency information ofthe analyzed specimen 60 (refer to FIG. 4). In steps S102 and S103 ofFIG. 3 and steps S202 and S203 of FIG. 6, the observer sets theparameters or sets the observational region 24. For example, thecollection of the specific spatial frequencies may represent the samestructure. If only the structure is intended to be observed, by settingthe spatial frequency of the structure intended to be observed throughthe parameter setting portion 23 of the display section 21, it ispossible to adjust the observational image of the specimen 60 inaccordance with the spatial frequency.

By detecting the information of the specimen 60 through theabove-mentioned method, the calculation section 20 is able toautomatically set the illumination shape appropriate for the observationof the specimen. However, various modifications can be further appliedto the examples.

For example, in the microscope system 200 shown in FIG. 11, by using twoimage sensors at the same time, it is possible to simultaneously acquiretwo information pieces of the image of the imaging plane and the imageof the pupil 273. However, by inserting a detachable relay lens betweenthe pupil 273 and the image sensor 80 and forming an image conjugate tothe pupil 273 on the image sensor 80, it is possible to acquire thespatial frequency information of the specimen 60 even when using only asingle image sensor.

Furthermore, the microscope system 200 may be configured such that aninterferometer is built in the microscope system 200 and theinterference image of the pupil is acquired. With such a configuration,by checking out the amplitude information of the pupil, it is alsopossible to acquire the phase information of the specimen 60. Theinterferometer can cause the object light, which is transmitted throughthe specimen 60, and the reference light, which is not transmittedthrough the specimen 60, to interfere with each other, measure theinterference image through the second image sensor 280, and obtainFourier spectrum of the object, thus it is possible to acquire the phaseinformation of the object. In the case of forming the interferometer, itis preferable that a laser or the like be used in the illumination lightsource 30. By using the laser, it is possible to obtain strongmonochromatic light, and thus it is possible to further reduce the sizeof the point light source. Furthermore, from the plurality ofillumination directions, the interference image between the diffractedlight of the object light and the reference light is detected by theimage sensor 80, whereby it is also possible to form three-dimensionalpicture of the specimen 60. A detailed description of the microscopeusing the interferometer is disclosed in, for example, WO 2008/123408.

Furthermore, the point light sources shown in FIG. 10B may be formed byusing a mask having point openings instead of the first spatial lightmodulation element 90.

Furthermore, a larger number of the point light sources shown in FIG.10B are formed along the outer peripheral portion of the circle 31.Thereby, it is possible to obtain the spatial frequency or thediffracted light in a case where the inclined illuminations from moredirections are applied to the specimen 60. Furthermore, the plurality ofpoint light sources shown in FIG. 10B may be measured at a plurality ofsingle wavelengths, for example, the respective wavelengths of red,blue, and green. When the respective point light sources shown in FIG.10B are measured at red, blue, and green, a description will be givenwith reference to FIG. 13C.

FIG. 13C is a diagram of the display section 21 which displays a pictureof an image of the pupil 273 in each wavelength of red, blue, and greendetected by the second image sensor 280 in a case where the specimen 60is a biological object. FIG. 13C is a diagram in the case where thespecimen 60 is measured by using the respective wavelengths of red,blue, and green through, for example, the point light source 32 b ofFIG. 10B. In actual measurement, the images of the pupil 273 formed byall the point light sources 32 a to 32 e are measured. In FIG. 13C, theimage 234 a which is detected at the wavelength of red, the image 234 bwhich is detected at the wavelength of blue, and the image 234 c whichis detected at the wavelength of green are displayed on the same screen.Since the spatial frequency is inversely proportional to the wavelength,by measuring the image of the specimen 60 in the pupil 273 for eachwavelength of light, it is possible to further accurately examine thespatial frequency distribution of the specimen 60. In FIG. 13C,existence of the structure, in which the region of the picture 234 adetected at the wavelength of red has a relatively small spatialfrequency and the region of the picture 234 b detected at the wavelengthof blue has a relatively large spatial frequency, is estimated.

SECOND EXAMPLE

In the first example, the microscope system 100 having the bright fieldmicroscope was described, and in the second example, a microscope system300 having the phase-contrast microscope will be described.

<Microscope System 300>

FIG. 14A is a schematic configuration diagram of the microscope system300. The microscope system 300 is an optical microscope system forobserving the specimen 60. The microscope system 300 mainly includes:the illumination light source 30; the illumination optical system 40;the imaging optical system 70; the image sensor 80; and the calculationsection 20. Furthermore, the illumination optical system 40 includes: afirst condenser lens; the wavelength filter 44; a first spatial lightmodulation element 390; and the second condenser lens 42. The imagingoptical system 70 includes the objective lens 71 and a second spatiallight modulation element 396. Furthermore, the stage 50 is disposedbetween the illumination optical system 40 and the imaging opticalsystem 70, and the specimen 60 is placed on the stage 50.

The second spatial light modulation element 396 is disposed at aposition of the pupil of the imaging optical system 70 or in thevicinity thereof. Furthermore, the first spatial light modulationelement 390 is disposed at the position conjugate to the pupil of theimaging optical system 70 in the illumination optical system 40. Thefirst spatial light modulation element 390 is an element which is ableto arbitrarily change the intensity distribution of the transmittedlight, and is constituted by a liquid crystal panel, a DMD, or the like.The second spatial light modulation element 396 is constituted by such aliquid crystal panel which is an element capable of changing the phase.Furthermore, it is preferable that the second spatial light modulationelement be configured to freely change the phase and the light intensitydistribution.

In FIG. 14A, the light, which is emitted from the illumination lightsource 30, is indicated by the dotted line. The illumination light LW31,which is emitted from the illumination light source 30, is convertedinto the light LW32 through the first condenser lens 41. The light LW32is incident to the first spatial light modulation element 390. The lightLW33, which is transmitted through the first spatial light modulationelement 390, is transmitted through the second condenser lens 42, isconverted into the light LW34, and propagates toward the specimen 60.The light LW35, which is transmitted through the specimen 60, istransmitted through the objective lens 71, is converted into the lightLW36, and is incident to the second spatial light modulation element396. The light LW36 is transmitted through the second spatial lightmodulation element 396, is converted into the light LW37, and forms animage on the image sensor 80. The output data of the picture formed onthe image sensor 80 is sent to the calculation section 20. Thecalculation section 20 calculates the illumination shape, which is mostappropriate to the specimen 60, on the basis of the output data of thepicture obtained from the image sensor 80, the shape data of thetransmission region (the illumination region) 391 formed by the firstspatial light modulation element 390, and the shape data of the secondspatial light modulation element 396. Then, the shape, which isappropriate for the observation of the specimen 60 subjected to thecalculation, is transmitted to the first spatial light modulationelement 390 and the second spatial light modulation element 396. Inaddition, when the wavelength filter 44 is provided, only light with aspecific wavelength is transmitted through the wavelength filter 44, andis incident to the first spatial light modulation element 390.

FIG. 14B is a top plan view of the first spatial light modulationelement 390. In the first spatial light modulation element 390, thelight transmission region (the illumination region) 391 is formed in aring shape, and a region other than the transmission region 391 isformed as a light blocking region 392.

FIG. 14C is a top plan view of the second spatial light modulationelement 396. Since a phase modulation region 397 is formed in a ringshape in the second spatial light modulation element 396, the phase ofthe light, which is transmitted through the phase modulation region 397,is shifted by ¼ wavelength. The phase of the light, which is transmittedthrough the diffracted light transmission region 398 as a region otherthan the phase modulation region 397, is unchanged. The phase modulationregion 397 is formed to be conjugate to the transmission region 391 ofthe first spatial light modulation element 390.

The 0-order light (the transmitted light) of the microscope system 300is transmitted through the first spatial light modulation element 390,is transmitted through the phase modulation region 397 of the secondspatial light modulation element 396, and reaches the image sensor 80.Furthermore, the diffracted light, which is emitted from the specimen60, is transmitted through the diffracted-light transmission region 398of the second spatial light modulation element 396, and reaches theimage sensor 80. Then, the 0-order light and the diffracted light forman image on the image sensor 80. Generally, the 0-order light has anintensity stronger than that of the diffracted light, and thus it ispreferable to form a filter for adjusting the intensity of the light ofthe phase modulation region 397. This filter can be formed by adding anoptical element which is capable of freely changing the spatialdistribution of the transmittance, or the like to the second spatiallight modulation element 396, wherein the optical element has an arrayof cells and can be electrically controlled (for example, PCT JapaneseTranslation Patent Publication No. 2010-507119).

The first spatial light modulation element 390 and the second spatiallight modulation element 396 are able to freely change the sizes and theshapes of the transmission region 391 and phase modulation region 397.For example, when the diameter of the transmission region 391 of thefirst spatial light modulation element 390 increases, the numericalaperture of the transmitted light increases, and thus it is possible toincrease the resolution. Furthermore, by using the method of derivingthe illumination shape shown in the first example, the shape of thetransmission region 391 of the first spatial light modulation element390 may be optimized. The ring-shaped region 397 of the second spatiallight modulation element 396 is always formed to be conjugate to thetransmission region 391 of the first spatial light modulation element390. Hence, it is preferable that the shapes of the transmission region391 and the ring-shaped region 397 be synchronously changed.

The best modes for carrying out the invention have hitherto beendescribed, but it will be readily apparent to those skilled in the artthat various modifications can be applied to the examples withoutdeparting from the technical scope of the invention.

What is claimed is:
 1. A microscope system as an optical microscopesystem for observing a specimen, the microscope system comprising: animaging optical system that forms an image of transmitted light orreflected light from the specimen; an illumination light source thatilluminates illumination light to the specimen; an illumination opticalsystem that has a first spatial light modulation element, which formsbeams of illumination light and which changes the intensity distributionof the illumination light at a conjugate position of a pupil of theimaging optical system, and illuminates light, which is originated fromthe illumination light source, on the specimen; an image sensor thatdetects light through the imaging optical system; and a calculationsection that initially performs an estimating process that estimatesinformation concerning the specimen in order to facilitate performing asubsequent observational process that calculates an optimum intensitydistribution of the illumination light for observation of the specimen,wherein the estimating process includes estimates of: (i) phaseinformation of the specimen, microscopic structure information, orinformation with respect to a wavelength of illumination light on thebasis of output data detected by the image sensor with two beams ofillumination light formed by the first spatial light modulation elementand of which coherence factors are different from each other, thecoherence factor (σ=NA′/NA) being a ratio of a numerical aperture NA′ ofthe illumination light formed by the first spatial light modulationelement to a numerical aperture NA of the imaging optical system; or(ii) phase information of the specimen or spatial frequency informationon the basis of the output data with two beams of illumination lightformed by the first spatial light modulation element and, each of whichis a monochromatic point light source where the coherence factor thereofis equal to 0, one of the two point light sources being disposed at anoptical axis and another one being disposed separate from the opticalaxis.
 2. The microscope system according to claim 1, wherein, in theestimating process, the first spatial light modulation element forms thetwo illumination light beams sequentially in a point shape and acircular shape larger than the point shape, and wherein the calculationsection calculates whether phase information is included in the specimenon the basis of the output data of the illumination light beam havingthe point shape and the illumination light beam having the circularshape.
 3. The microscope system according to claim 1, wherein, in theestimating process, the first spatial light modulation element forms thetwo illumination light beams sequentially in a point shape and anannular shape, and wherein the calculation section calculates whether amicroscopic structure is included in the specimen on the basis of theoutput data of the illumination light beam having the point shape andthe illumination light beam having the annular shape.
 4. The microscopesystem according to claim 1, wherein the first spatial light modulationelement is constituted by a plurality of movable reflection mirrors. 5.The microscope system according to claim 1, wherein the first spatiallight modulation element is constituted by an optical element having aplurality of spaces, each of which has a variable transmittance.
 6. Themicroscope system according to claim 1, wherein, in the estimatingprocess, the calculation section calculates a spatial frequency or acontrast of the specimen on the basis of the output data detected by theimage sensor.
 7. The microscope system according to claim 6, wherein, inthe estimating process, the calculation section sets an observationalregion of the specimen on the basis of the contrast or the spatialfrequency.
 8. The microscope system according to claim 1, wherein, inthe estimating process, a wavelength of the illumination light, which isilluminated on the specimen, is changed, and wherein the calculationsection estimates a wavelength for the observation of the specimen onthe basis of the output data for each of the changed wavelengths.
 9. Themicroscope system according to claim 1, further comprising: a displaysection that displays a parameter setting portion that sets a parameterfor inputting an observation condition, which is requested and allowedby an observer, for an observational image of the specimen and a regionsetting portion which sets an observational region of the observationalimage.
 10. The microscope system according to claim 9, wherein theparameter setting portion is able to set at least one of theillumination shapes and sizes, a spatial frequency band which isintended to be mainly observed, a resolution of the set observationalregion, and a contrast thereof.
 11. The microscope system according toclaim 1, wherein the image sensor is disposed at the pupil of theimaging optical system or at the conjugate position of the pupil, anddetects output data of a picture of the pupil of the specimen under theillumination condition of a monochromatic point light source, andwherein the calculation section calculates the optimum intensitydistribution of the illumination light for observation from theillumination condition and the output data of the picture of the pupil.12. The microscope system according to claim 11, wherein the calculationsection obtains spatial frequency information of the specimen from theoutput data of the picture of the pupil, and calculates a periodicstructure of the specimen.
 13. The microscope system according to claim11, wherein a wavelength of a monochromatic point light source is set toa value designated by the observer.
 14. The microscope system accordingto claim 11, wherein the microscope system is adapted to change awavelength of a monochromatic point light source to another wavelengthso that a plurality of wavelengths are selected, and the output data ofa picture of the pupil of the specimen is detected for each wavelength.15. The microscope system according to claim 11, wherein the point lightsource is embodied by using a mask having a point opening.
 16. Themicroscope system according to claim 1, wherein the calculating of theoptimum intensity distribution of the illumination light for observationof the specimen involves selection of an illumination shape of anillumination region of the first spatial light modulation element.